Flow cytometry is a sensitive and versatile probe of the optical characteristics of microscopic particles, with widespread applications including hematology, immunology, genetics, food science, pharmacology, microbiology, parasitology, and oncology. Optical flow cytometers use light scattering and fluorescence to determine physical and chemical properties of the particles. For measurement, particles are arranged in single file, typically by hydrodynamic focusing within a sheath fluid, and interrogated by a light beam propagating orthogonal to the flow axis. Flow cytometers often use two concentric fluids to carry particles through the measurement zone, where optical measurement occurs. The use of two concentric fluids facilitates the passage of the particles through the measurement zone in a single file fashion, and helps avoid clogging of the flow channel. Hydrodynamic focusing is a phenomenon that leads to a single file flow of particles as a result of the very small dimensions of the flow channel. A sample is injected into a flowing sheath fluid; the dimensions of the flow channel become more narrow, causing the dimensions of the stream of sample to become more narrow also. FIG. 1 is a cross section of flow in an art-known sheath flow accomplished by injecting, via a needle or other concentric opening, a center fluid (41) containing a sample with particles (42) into a sheath fluid (40), surrounded by air. Hydrodynamic focusing requires laminar flow of the fluids; any turbulence would cause mixing of the concentric fluids. The optical properties of the particles are measured in the measurement zone. Scattered light and fluorescence are measured by two or more photodetectors positioned around the illuminated portion of the flow stream. A first photodetector can be positioned to collect small angle scattering. A second photodetector is often positioned at about 90.degree. to the forward scattering direction to collect large angle scattering and fluorescence.
Existing commercial cytometers are large and complicated instruments requiring skilled operators. To increase the accessibility of flow cytometry, compact cytometers are desired.
The flow behavior of liquids at the microscopic level is significantly different from the flow behavior of liquids at the macroscopic level. In microstructures, i.e. microfabricated fluidic devices, practically all flow is laminar, as a result of the extremely small channel diameters. Laminar flow allows two or more fluids to flow parallel to each other without turbulence-induced mixing. However, because the channel diameters are very small, diffusion is significant. Since diffusion occurs in all directions, a component of one layer may diffuse to another layer, perpendicular to the direction of flow.
Sheath flow is a particular type of laminar flow in which one layer is surrounded by another layer on more than one side. Concentric layers of fluids, that is, where one layer is completely surrounded on all sides by another layer, is one example of sheath flow. Sheath flow is useful because it positions particles with respect to illuminating light, e.g., a laser beam, and it prevents particles in the center fluid, which is surrounded by the sheath fluid, from touching the sides of the flow channel and thereby prevents clogging of the channel. Sheath flow allows for faster flow velocities and higher through-put of sample material. Faster flow velocity is possible without shredding cells in the center fluid because the sheath fluid protects the cells from shear forces at the walls of the flow channel. Sheath flow is useful in many applications, including but not limited to, any application in which it is preferable to protect particles by a layer of fluid, for example in applications wherein it is necessary to protect particles from air. Other applications include flow cytometry and combustion processes wherein an inner core layer burns at a different temperature from that of an outer layer. In the latter application, the sheath flow module of this invention can be used to create a flame of two or more combustible fluids with the outer sheath fluid burning, for example, at a higher temperature than the inner core fluid. In this case, the outer sheath fluid can be used to heat the inner core fluid. Of course, the temperatures of the fluids can be reversed, i.e., the outer sheath fluid can be one which burns at a lower temperature than the inner core fluid. Control of flame shape or color is possible using the sheath flow module of this invention.
In a microfabricated flow channel, a challenge is to focus light into the channel and to collect near forward scattered and high angled scattered light out of the channel. A few microfabricated flow cytometer flow channels have been reported. Miyake et al. [Proceedings of the IEEE Micro Electro Mechanical Systems Workshop, pp. 265-270, Nara, Japan, January 1991] describes a micromachined sheath flow channel made of five stacked plates. Three metal plates are used to create a flow having a sample core within a sheath, and glass plates on the top and bottom of the stack provide optical access to the flow channel for illumination through the top and forward scattered light collection through the bottom. The top and bottom plates provide a sheath fluid inlet. The middle plate provides for the sample inlet in the center, with the sheath fluid inlets on both sides. It appears that 90.degree. scattering cannot be collected. Sobek et al. [Proceedings of the IEEE Micro Electro Mechanical Systems Workshop, pp. 219-224, Fort Lauderdale, Fla., February 1993] describes a four-layer silicon microfabricated hexagonal sheath flow channel. The channel is formed between two of the silicon wafers. Integrated optical waveguides intersecting the channel are used to couple laser light into the channel and out of the channel in the forward direction. At this intersection, the top and bottom walls of the channel are silicon nitride/silicon dioxide windows for 90.degree. light collection. Each window is fabricated by growing an oxide layer on a silicon wafer, bonding the oxide layer to a second silicon wafer, etching away the silicon on both sides of the oxide at the window region and depositing a nitride layer. Sheath flow with a sample in the center of the sheath stream is accomplished by injecting sample via a hypodermic needle into the center of the stream of sheath fluid. Sobek et al. [Proceedings of the Solid-State Sensors and Actuators Workshop, Hilton Head, S.C., June 1994] describes a sheath flow channel fabricated between two fused silica wafers. To couple light into the channel and out in the forward direction, optical fibers are sandwiched between the wafers orthogonal to the flow axis. Fluorescence is collected through the upper transparent wafer. Again, sheath flow is accomplished by injection of the sample via a hypodermic needle into the center of the sheath stream.
U.S. Pat. No. 5,726,751, "Silicon Microchannel Optical Flow Cytometer," issued Mar. 10, 1998, which is incorporated by reference herein in its entirety, discloses a flow cytometer comprising a v-groove flow channel formed by micromachining a silicon wafer. This reference describes a flow cytometer made of two components: a flow cytometer optical head and a disposable flow module. The flow module of this reference exploits the fact that anisotropic etching of single crystalline silicon wafers provides access to reflective surfaces with precisely etched angles relative to the surface of the wafer. The facets are used for reflecting, as opposed to transmitting, an illuminating laser beam. This reference suggests the use of a sheath flow in a v-groove but does not teach a novel method or apparatus for achieving sheath flow.